Streptavidin-binding peptide

ABSTRACT

The invention relates to novel streptavidin binding peptides.

FIELD OF THE INVENTION

The invention relates to a streptavidin-binding peptide and to methodsfor the production of a streptavidin-binding peptide in a cell-based orcell-free protein biosynthesis system. Further, the invention relates tothe use of a streptavidin-binding peptide for purifying a definedprotein produced in a protein biosynthesis system, and to the use of astreptavidin-binding peptide for marking a defined protein.

PRIOR ART

Methods for efficiently expressing defined proteins in various pro- andeukaryotic organisms are known in the art and do not require furtherexplanations. As defined proteins are understood here peptides and/orproteins, which are expressed naturally or after transformation or useof defined RNA in the organism used for the expression or cell-freeexpression system and are enriched in purification steps.

Methods for the cell-free expression of defined proteins are, forinstance, known in the art from documents EP 0312 617 B1, EP 0401 369 B1and EP 0593 757 B1. According thereto, the components necessary fortranscription and/or translation are incubated, in addition to a nucleicacid strand coding for a defined protein, in a reaction vessel and afterthe expression the polypeptides/proteins are isolated from the reactionsolution. The components necessary for transcription as well as fortranslation can be obtained from the supernatants of pro- or eukaryoticcell lysates after centrifugation.

An essential problem for the expression of defined proteins in pro- andeukaryotic organisms and for the cell-free expression is thepurification and/or the detection of the expressed defined proteins.This is particularly problematic with defined proteins, for which thereare no antisera or monoclonal antibodies. For simplifying thepurification and the detection of such defined proteins, they areexpressed as so-called fusion proteins. Hereby, an amino acid sequenceis added N- and/or C-terminally to the defined protein or insertedbetween two protein domains (internally), the fusion partner. Thishappens on the nucleic acid level, so that the defined protein as wellas the fusion partner added for the detection and/or purification areexpressed together during a transcription/translation process as achimeric (fusion) protein, consisting of the defined protein and thefusion partner. The mentioned fusion partner may be a single amino acid,but also a peptide or protein. For these added fusion partners, thereare available binding partners immobilized for purification, and thefusion proteins can be isolated with these binding partners. In additionto the possibility of the purification of the proteins, they may also bedetected with binding partners being specific for the fusion partner.These binding partners may be antibodies specific for the fusion partneror also other proteins, peptides or chemical compounds specificallybinding to the fusion partner.

Examples for such fusion partners are the Myc-tag (Munro & Pelham, Cell46:291-300, 1986; Ward et al., Nature 341:544-546, 1998), the FLAGpeptide (Hopp et al., Bio/Technology 6:1204-1210, 1988), the KT3 epitopepeptide (Martinet et al., Cell 63:843-849, 1990; Martin et al., Science255:192-194, 1992) and the alpha-tubulin epitope peptide (Skinner etal., J. Biol. Chem. 266:14163-14166, 1991), all of which weresuccessfully used for the detection and in part also for thepurification of proteins. For part of the fusion partners normallyhaving a length of 3 to 12 amino acids, it could be shown that they donot affect the biological function of the defined proteins. Thebiological function of the defined protein is, however, affected withincreasing length of the fusion partner, since the additionallyexpressed amino acids can affect e.g. the development of the secondary,tertiary and/or quaternary structure. Longer fusion partners are thussuitable for the detection of the proteins, but less for thepurification of the proteins. On the other hand, longer fusion partnersfrequently have a higher affinity with their specific binding partners.

A substantial drawback of the above fusion partners with respect to thepurification is that the binding to the binding partner is based on anantigen/antibody binding, and the production and purification of theantibodies used as binding partners is time-consuming and expensive.Another drawback is that the antigen/antibody binding requires a verystrong binding between the binding partner, e.g. antibody immobilized ata matrix, and the fusion partner. This has as a consequence that for theelution of the fusion protein bound via the fusion partner, sometimesextremely “unphysiological” conditions with respect to the definedprotein, are generated. Unphysiological conditions are, e.g., very highor extremely low salt concentrations, use of chaotropic salts and pHvalues differing by far from the natural pH value of the definedprotein. This may affect, under certain circumstances, the structureand/or the functionality of the defined protein or irreversibly destroyit. Correspondingly, the purification of the defined proteins shouldtake place under as “gentle” and physiologic conditions as possible, inorder to maintain the functionality of the proteins. For three of theabove fusion partners (Hopp et al., 1988; Martin et al., 1990; Skinneret al., 1991), an elution could even be achieved under gentle conditionsby means of competitive peptides, however there is still the problem ofthe time-consuming and expensive antibodies to be purified (and servingas binding partners) and the binding thereof to the matrix.

Further fusion partners consisting of 8 to 9 amino acids are known inthe art from the documents U.S. Pat. No. 5,506,121 and Schmidt & Skerra(Protein Engineering, vol. 6, no. 1, 109-122, 1993). The fusion partnersdisclosed therein are capable of binding to the streptavidin or to the“core” streptavidin, a proteolytically cleaved product of thestreptavidin (Bayer et al., Biochem. J. 259:369-376, 1989).

All known Fusion partners that bind to the streptavidin containing theamino acid sequence HPQ, the so-called HPQ binding motif coming intointeraction with the biotin-binding pocket of the streptavidin. To thealready known peptides belong the so-called Strep-tag I: AWRHPQFGG [SEQID NO: 18] with a dissociation constant of Kd of 10-37 μM, the so-calledStrep-tag II: WSHPQFEK [SEQ ID NO: 19] with a dissociation constant Kdof 18-72 μM, and the so-called SBP-tag: MDEKTTGWRGGIIVVEGLAGELEQLRARLEHHPQGQREP [SEQ ID NO: 20] with a dissociation constant Kd of 2.5 nM. Incontrast to the two short Strep-tags I and II, the longer SBP-tag has asubstantially stronger binding to the streptavidin. However, asmentioned above, the function of the defined protein is particularlyaffected with increasing length of the fusion partner. Further,particularly long fusion partners disturb the crystallization of thedefined proteins.

TECHNICAL OBJECT OF THE INVENTION

It is the technical object of the invention to provide astreptavidin-binding peptide that is as short as possible yet still hasa strong binding affinity to streptavidin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a SDS polyacrylamideelectrophoresis gel showing the result of purification of FABP withstreptavidin-binding peptide according to motif 3 as a fusion partnerafter cell-free protein biosynthesis via a streptavidin affinity column.

FIG. 2 is a schematic representation of a SDS polyacrylamideelectrophoresis gel showing the result of purification of FABP withstreptavidin-binding peptide according to motif 3 as a fusion partnerafter cell-free protein biosynthesis via a StrepTactin affinity column.

BASICS OF THE INVENTION AND PREFERRED EMBODIMENTS

For achieving the technical object, the invention relates to astreptavidin-binding peptide comprising or consisting of an amino acidsequence according to SEQ ID NOS: 1-12. By the streptavidin-bindingpeptide according to the invention comprising an amino acid sequenceaccording to SEQ ID NOS: 1-12 it is achieved, compared to prior art, asubstantially stronger binding between the streptavidin-binding peptideand the streptavidin, or the streptavidin-binding peptide cansubstantially be shorter with the same binding strength.

Further, the invention relates to a nucleic acid coding for astreptavidin-binding peptide according to SEQ ID NOS: 1-12 and a plasmidcomprising a nucleic acid according to the invention. It is understoodthat the nucleic acid coding for a streptavidin-binding peptideaccording to the invention and/or the plasmid can be adjusted to therespective expression system/protein biosynthesis system. The plasmidmay be adapted as an expression vector, in particular for bacteria,comprising a region with at least one interface for a restrictionenzyme, in which the nucleic acid sequence coding for a defined proteinmay be inserted, and thus the defined protein is expressed together withthe streptavidin-binding peptide according to SEQ ID NOS: 1-12. It isunderstood that the region coding for the defined protein and for thestreptavidin-binding peptide according to SEQ ID NOS: 1-12 are under thecontrol of a suitable promoter and/or operator and terminator. Theregion with at least one interface for at least one restriction enzymemay lie in the 5′ as well as 3′ direction of the nucleic acid regioncoding for the streptavidin-binding peptide according to SEQ ID NOS:1-12. The nucleic acid region coding for the streptavidin-bindingpeptide according to SEQ ID NOS: 1-12 needs not to follow immediately tothe nucleic acid region coding for the defined protein, rather there maybe nucleic acids coding for 1 to 20 amino acids, in particular for 5 to10 amino acids, between the two regions.

The invention also relates to a method for the production of astreptavidin-binding peptide according to SEQ ID NOS: 1-12, wherein anucleic acid is expressed or overexpressed in a cell-based or cell-freeprotein biosynthesis system. The thus produced peptide can easily beisolated via the binding to the streptavidin. The obtained translationproduct, i.e. a streptavidin-binding peptide according to SEQ ID NOS:1-12, is contacted with immobilized streptavidin and is bound thereto.After separation of the solution with substances not bound tostreptavidin, the translation product is eluted. As an elution agent,buffers can be used, which contain biotin or related substances and/orderivatives, such as iminobiotin, desthiobiotin and/or diaminobiotin.The obtained streptavidin-binding peptide according to SEQ ID NOS: 1-12carries in the case of the fusion with the defined protein this protein.It may also be used independently from a defined protein for theantibody production. The obtained antibodies can be used e.g. for thedetection or purification of the streptavidin-binding peptides accordingto SEQ ID NOS: 1-12 or in the case that the streptavidin-binding peptideaccording to SEQ ID NOS: 1-12 is employed as a fusion partner, of thedefined protein bound to this fusion partner.

It is understood that the production of such a streptavidin-bindingpeptide containing an amino acid sequence according to SEQ ID NOS: 1-12is also possible by means of chemical solid phase synthesis, forinstance with an automatic synthesis system made by Abimed (Langenfeld,Germany). This method is based on the standard protocols of the Fmocchemistry (Fmoc=9-fluorenylmethyloxycarbonyl).

The invention also relates to the use of a streptavidin-binding peptideaccording to SEQ ID NOS: 1-12 for the purification of a defined proteinproduced in a protein biosynthesis system, wherein a nucleic acid codingfor the protein and, connected therewith, for the streptavidin-bindingpeptide is subjected to a transcription and/or translation, wherein asolution comprising the thus obtained translation product is contactedwith immobilized streptavidin and is bound thereto, and wherein afterseparation of the solution with substances not bound to streptavidin thetranslation product is eluted. The elution may take place under gentleconditions for the defined protein. As an elution agent, buffers can beused, which contain biotin or related substances and/or derivatives,such as iminobiotin, desthiobiotin and/or diaminobiotin. The produceddefined protein may comprise, N and/or C terminally, thestreptavidin-binding peptide according to SEQ ID NOS: 1-12 serving as afusion partner.

If a streptavidin-Sepharose column is used, the defined protein to beinvestigated can be immobilized at the matrix by means of thestreptavidin-binding peptide according to SEQ ID NOS: 1-12 serving as afusion partner and isolated from a mixture of molecules, e.g. a celllysate.

The invention also relates to the use of a streptavidin-binding peptideaccording to SEQ ID NOS: 1-12 for labeling a defined protein, wherein anucleic acid coding for the protein and, connected therewith, for thestreptavidin-binding peptide is subjected to transcription and/ortranslation, wherein the thus obtained translation product is contactedwith a streptavidin conjugate comprising a reporter molecule and boundthereto. The labeled defined protein may comprise, N and/or Cterminally, the streptavidin-binding peptide according to SEQ ID NOS:1-12 serving for labeling. Reporter molecules may, for instance, beradioactive and/or radioactively labeled substances. It is understoodthat the streptavidin as such may also be radioactive and/orradioactively labeled; in this case a reporter molecule is not needed.Reporter molecules may also be fluorescent substances or enzymes, suchas alkaline phosphatase or peroxidase. Such streptavidin compoundscoupled to a reporter molecule, for instance proteins, which have thestreptavidin-binding peptide according to SEQ ID NOS: 1-12 as a fusionpartner, can be detected and/or quantified on a Western blot or in theELISA (enzyme linked immunosorbent assay). If the defined proteins arebinding proteins, other proteins binding thereto can also be detected inthis way. Binding proteins are in this context proteins, whichthemselves bind other proteins and/or themselves bind to other proteins,such as in an antigen/antibody binding or in a binding of a protein to areceptor.

Preferred is a streptavidin-binding peptide comprising less than 30amino acids, preferably less than 20 amino acids, most preferably lessthan 10 amino acids.

In the following, the invention is explained in more detail, based onfigures and examples representing embodiments only.

FIG. 1: purification of FABP with streptavidin-binding peptide accordingto motif 3 as a fusion partner after cell-free protein biosynthesis viaa streptavidin affinity column. A comparable amount of each fraction wasanalyzed by means of SDS polyacrylamide gel electrophoresis. (A)Coomassie stained and (B) autoradiogramme. The samples in the numberedtracks are (1) molecular weight markers; (2) reaction mixture; (3)passage of the sample application; (4-6) washing fractions; (7-9)elution fractions; (10) radioactive molecular weight marker.

FIG. 2: purification of FABP with streptavidin-binding peptide accordingto motif 3 as a fusion partner after cell-free protein biosynthesis viaa StrepTactin affinity column. A comparable amount of each fraction wasanalyzed by means of SDS polyacrylamide gel electrophoresis. (A)Coomassie stained and (B) autoradiogramme. The samples in the numberedtracks are (1) molecular weight markers; (2) passage of the sampleapplication; (3-5) washing fractions; (6-10) elution fractions; (11)radioactive molecular weight marker.

Example 1 Binding Peptides According to the Invention and ComparisonPeptides with Dissociation Constants

Using standard methods for this technology, the FAB protein (fatty acidbinding protein) (FABP) from bovine hearts was expressed in vitro in acell-free protein biosynthesis by means of a coupledtranscription/translation system. The expressed FAB protein had at the Nterminus an additional streptavidin-binding peptide composed of 15 aminoacids with different amino acid sequences as fusion partners.

Two binding peptides contain in their amino acid sequence the HPQ motif(motif 1 and 2) described in the prior art, and two of them contain thestreptavidin-binding peptide according to SEQ ID NOS: 1-6 according tothe invention (motif 3 and 4). The sequences of the peptides are shownin Table 1. In addition, the expressed proteins were provided at the Cterminus with a “His-tag” consisting of 6 histidines. The proteins werepurified by means of affinity chromatography via Ni2+IDA-agarose usingstandard methods. It can be seen that with equal length, bindingpeptides of a sequence according to the invention have a substantiallylower dissociation constant than a binding peptide with HPQ motif. TheKd value of a binding peptide according to the invention is in the orderof the SBP-tag in spite of the 2.5 fold length of the SBP-tag.

TABLE 1 Dissociation Sequence constant [kd] motif 1D L Y D I D R N W V G H P Q G   8 μM SEQ ID NO: 14 motif 2D N Y D A D L A W D T H P Q D  70 μM SEQ ID NO: 15 motif 3D V E A W L D E R V P L V E T  84 nM SEQ ID NO: 16 motif 4D V E A W I A D P A V H F T T 200 nM SEQ ID NO: 17

Example 2 Kind of Measurement of the Dissociation Constant of Table 1

The measurements of the dissociation constant were performed with aBiacoreX system and the sensor chip NTA of the company Biacore. Theproteins generated during the expression described in Example 1 wereinvestigated, i.e. FAB proteins, which had at the N terminus a peptideconsisting of 15 amino acids as fusion partners and at the C terminus 6histidines, which were needed for the immobilization on the sensor chip.The binding affinity of the proteins generated during the expression tostreptavidin described in Example 1 was measured in the Biacore deviceaccording to manufacturer's instructions. The protein to be investigatedis on the sensor chip, and a streptavidin solution having a definedconcentration is sprayed in. The interaction (binding) between the twomolecules is measured by the device and quantified as so-calledresonance units (RU). For the measurement, the buffers specified by themanufacturer were used.

The results of the measurements are shown in Table 2. The obtainedmeasurements were evaluated with the provided software and led to thedissociation constants shown in Table 1.

TABLE 2 Motif 1 2 3 4 Streptavidin conc. RU RU RU RU 15 nM  98 30 nM 242 68 60 nM 461 171 125 nM 613 280 250 nM 704 384 500 nM  62 786 478 1 μM123 — 560 2 μM 233 946 644 3 μM —  64 — 4 μM 407 — 983 6 μM —  98 8 μM621 — 15 μM — 201 16 μM 779 — 30 μM — 337 32 μM 955 — 60 μM 536

Example 3 Purification of a Fusion Protein

Using standard methods, the FAB protein having at the N terminus anadditional peptide consisting of 15 amino acids with the amino acidsequence DVEAWLDERVPLVET [SEQ ID NO: 16] (motif 3) as fusion partners,is expressed in vitro in a cell-free protein biosynthesis by means of acoupled transcription/translation system. For purification of theoverexpressed defined protein, the streptavidin was coupled to a solidphase. As a solid phase was used a Sepharose. The expressed FAB proteinwas then purified using standard methods via a column comprisingstreptavidin-Sepharose or StrepTactin-Sepharose. For the purificationthe following buffers were used: washing buffer (100 mM Tris-HCl (pH8.0), 150 mM NaCl, 1 mM EDTA).

For the elution of streptavidin, the washing buffer contained 2 mMbiotin, and for the elution of StrepTactin, the washing buffer contained2.5 mM desthiobiotin.

The percentage distribution of the used defined protein on the variousfractions of the affinity chromatography can be taken from Table 3. InTable 3, D represents the application of sample/passage, W washingfraction, and E elution fraction.

TABLE 3 Fraction D W1 W2 W3 E1 E2 E3 E4 E5 Tot. Streptavidin 3.5% 6.8%1.0% 0.3% 0.4% 76.9% 0.7% — — 89.6% StrepTactin 3.0% 7.4% 1.8% 0.9% 0.9%45.9% 23.2% 1.0% 0.3% 84.4%

When using streptavidin-Sepharose, 90% of the applied protein could berecovered from the column, 78% for the eluate. The quality of thepurification is shown in FIG. 1.

When using StrepTactin-Sepharose, 84% of the applied protein could berecovered from the column, 71% for the eluate. The quality of thepurification is shown in FIG. 2.

Example 4 Optimization of Individual Binding Peptides

The binding peptide (FAx 3) with the sequence DVEAWLDERVPLVET [SEQ IDNO: 16] was subjected to a substitution analysis, and it was intendedthereby to identify a possible minimum motif (=shortened peptide) andexamine the effect of individual amino acid exchanges on the bindingspecificity. In a substitution analysis, peptides are synthesized bymeans of spot methods (Frank, R. 1992) on a cellulose membrane servingas a solid phase. Every position of the peptide comprising 15 aminoacids was systematically replaced by the other 19 L amino acids.

The substitution analysis was incubated with a peroxidase-labeledstreptavidin and developed with a luminescence substrate. Thereby it waspossible to find out, how far the peptide can be shortened and whichamino acids are “conserved” and thus not or only very poorlyexchangeable. The following picture resulted: a peptide, which hassufficient streptavidin binding, is the 6-mer peptide DVEAWL [SEQ ID NO:16]. A clearly better streptavidin binding has the 9-mer peptideDVEAWLDER [SEQ ID NO: 16]. The substitution analysis also indicatedpositions, where the exchange of the original amino acid by another onecould result in an improvement of the streptavidin binding.

The information supplied by the substitution analysis was verified bydetermining the binding constants of different peptides by surfaceplasmon resonance spectroscopy using a BIAcore device. The measurementswere made with the NTA sensor chip of the company BIAcore and by meansof the fatty acid-binding protein from bovine hearts (FABP). The FABPserved for the immobilization and presentation of the peptide. Further,it was intended to thus simulate the conditions occurring in an affinitychromatography, since the peptide is to be used in the future as anaffinity peptide for the purification of proteins. In contrast to thefirst measurements, where the peptides to be verified replaced the fourN terminal amino acids of the FABP, the arrangement of the peptides wasthis time also N terminal, but they were not part of the FABP. TheHis-tag necessary for the immobilization was also modified. It waslocated same as for the first measurements at the C terminus of theFABP, was however extended from six to eight histidines and furtherspaced by a linker consisting of two glycines from the FABP. Thereby,the immobilization of the ligands could distinctly be improved, and thusthe drift of the base line resulting from the washing-off of the ligandcould be minimized. For every measurement, an identical amount of therespective FABPs was immobilized, what corresponded to a rise of thebase line by 1,100 resonance units (RU). As a reference was used theFABP with the His-tag, but without N terminal binding peptide.

The binding constants obtained with the optimized measuring method werethe following:

Kd Peptide (FAx 3): D V E A W L D E R V P L V E T SEQ ID NO: 16  3.6 ±0.6 nM (AspValGluAlaTrpLeuAspGluArgValProLeuValGluThr) Shortened peptide(9-mer): DVEAWLDER 240 ± 40 nM SEQ ID NO: 16 Influence of other aminoacids on the shortened 9-mer peptide: 3 Asp: DVDAWLDER SEQ ID NO: 16 940± 140 nM 3 Gly: DVGAWLDER SEQ ID NO: 16 570 ± 90 nM 6 Phe: DVEAWFDER SEQID NO: 16 not measurable 6 Arg: DVEAWRDER SEQ ID NO: 16 not measurable 7Gly: DVEAWLGER SEQ ID NO: 16  17 ± 4 nM 8 Ala: DVEAWLDAR SEQ ID NO: 16160 ± 30 nM

A positive influence had in particular glycine at position 7 and alanineat position 8. Combination of these two amino acids:

Shortened peptide: DVEAWLGAR [SEQ ID NO: 16] 17±4 nM

Original peptide with Gly7: DVEAWLGERVPLVET [SEQ ID NO: 16] 4.2±0.7 nM

Original peptide: DVEAWLGARVPLVET [SEQ ID NO: 16] 4.1±0.6 nM

The original 15-mer peptide cannot further be improved. The shortened9-mer peptide however can distinctly be improved by a glycine in lieu ofthe asparagic acid at position 7. The alanine in lieu of the glutamineacid at position 8 also has a slightly better binding affinity withregard to streptavidin. By the combination of these two amino acids thebinding affinity cannot further be improved, however it will not becomeworse either.

The invention thus also comprises a 9-mer peptide with its not or onlyslightly conserved positions:

DVXAWLXXR (X=an arbitrary amino acid) [SEQ ID NO: 11]

Further, of course the shortened peptide with glycine at position 7:

DVEAWLGER [SEQ ID NO: 11]

and the shortened peptide with glycine at position 7 and alanine atposition 8:

DVEAWLGAR [SEQ ID NO: 11]

Example 5 Variants of Sequences According to the Invention

In the following, particularly suited sequences according to theinvention are given.

SEQ ID NO: 1: DVEAW SEQ ID NO: 2: DVEA SEQ ID NO: 3: VEAW SEQ ID NO: 4:DVE SEQ ID NO: 5: VEA SEQ ID NO: 6: EAW SEQ ID NO: 7: DVXAWSEQ ID NO: 8: DVXAWL SEQ ID NO: 9: DVXAWLX SEQ ID NO: 10: DVXAWLXXSEQ ID NO: 11: DVXAWLXXR SEQ ID NO: 12: DVXAWLXXRVPLVET SEQ ID NO: 13:VPLVET

X at position 3 may be arbitrary, however in particular E, D or G. X atposition 7 may be arbitrary, however in particular G or D. X at position8 may be arbitrary, however in particular A or E. Sequence 13 is anoptional carboxy-terminal extension of the sequence 11, and fromsequence 13 1 to 6 amino acids may be connected, beginningamino-terminally.

1. A purified streptavidin-binding peptide comprising or consisting ofan amino acid sequence according to SEQ ID NO: 11, wherein X at position3 is selected from the group consisting of glutamic acid, aspartic acid,and glycine.